CN112920976B - Virulent strain of mycoplasma ovipneumoniae and application thereof - Google Patents
Virulent strain of mycoplasma ovipneumoniae and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
The invention discloses a Mycoplasma ovipneumoniae virulent strain and application thereof, wherein the Mycoplasma ovipneumoniae is classified and named as Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) NJ01 strain, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020907, the preservation date is 12 months and 14 days in 2020. The application is the application of the mycoplasma ovipneumoniae in infection morbidity and preparation of the medicine for treating the mycoplasma ovipneumoniae. The mycoplasma culture medium adopted for culturing the mycoplasma ovipneumoniae virulent strain is simple and convenient to prepare, low in cost, quick in mycoplasma culture color change and high in titer.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a mycoplasma ovipneumoniae virulent strain and application thereof in establishing a mycoplasma ovipneumoniae infection morbidity model.
Technical Field
Mycoplasma ovipneumoniae (Mycoplasma pneumoniae of sheep and coat, MPSG) is a chronic respiratory infectious disease of sheep and goats caused by Mycoplasma ovipneumaniae (Mo). The disease is mainly characterized by asthma as a clinical characteristic, is widely prevalent in the world, is relatively susceptible to goats and sheep and almost universally exists, and causes great loss to the sheep raising industry. Establishing a mycoplasma ovipneumoniae infection pathogenesis model is an important precondition for researching the mycoplasma ovipneumoniae infection pathogenesis. Because mycoplasma is difficult to culture and mycoplasma ovipneumoniae is weak in virulence and difficult to attack, difficulty is brought to establishment of an infection attack model, and research on the disease is severely restricted.
In 23 pages of the literature, "bayongping, research on isolation and identification of mycoplasma ovipneumoniae and sensitive drug test, university of agriculture in Sichuan, 2011", it is reported that the growth rate of mycoplasma ovipneumoniae isolates increases with the increase of the number of passages, and is shortened from about the first 10 days to 3-5 days. As can be seen from the above reports, the mycoplasma ovipneumoniae has strict requirements on nutrition, is difficult to culture, and has slow growth and color change speed, which requires 3-10 days.
The vaccine research literature reports that the inactivated vaccine prepared by infecting pathogenic lung tissues, pleural effusion and the like of sheep with mycoplasma ovipneumoniae has certain effect on prevention and control; the treated mycoplasma culture is added into the tissue to prepare the inactivated vaccine, and the result shows that the inactivated vaccine has effective protection on the mycoplasma ovis pneumonia. However, the tissue inactivated vaccine is easy to be polluted by bacteria because of using the lung tissue of sheep disease, and the quality is difficult to control. After the proliferation of mycoplasma is reported in the relevant documents of subsequent vaccine research, the growth titer is determined and ultrafiltration concentration is carried out, so that the concentration of mycoplasma ovipneumoniae bacterial liquid reaches 6.0 x 10 8 -10.0×10 8 CCU/mL, then carrying out seedling preparation, and having better immune protection effect. However, the antigen needs to be concentrated in the preparation of the vaccine, so that the preparation period of the vaccine is long, the cost is high, the concentration of the hybrid protein is increased, and the production and the application of the vaccine are influenced. Therefore, there is an urgent need for strains and processes that can produce vaccines in pure culture without the need for concentration to produce vaccines with good protective efficacy to meet market demand.
Disclosure of Invention
The invention aims to: the technical problem to be solved by the invention is to provide a virulent strain of mycoplasma pneumoniae of sheep aiming at the defects of the prior art.
The invention also aims to solve the technical problem of providing the application of the mycoplasma ovipneumoniae virulent strain in establishing a mycoplasma ovipneumoniae infection pathogenesis model.
The invention finally solves the technical problem of providing the application of the mycoplasma ovipneumoniae virulent strain in preparing the medicine for treating diseases caused by mycoplasma ovipneumoniae infection.
In order to solve the first technical problem, the invention discloses a virulent strain of Mycoplasma ovipneumoniae, which is classified and named as Mycoplasma ovipneumoniae (Mycoplasma ovipneumaniae) NJ01 strain and is deposited in China center for type culture Collection, and the address is as follows: wuhan, wuhan university, zip code 430072, deposit number: CCTCC NO: m2020907. The strain is prepared by the inventor through self-collecting pathological materials in a certain sheep farm in Nanjing in 2018, and separating, purifying and identifying the pathological materials to obtain the mycoplasma ovipneumoniae.
In some embodiments, the heat stress protein 70 of mycoplasma ovipneumoniae has a similarity to other mycoplasma ovipneumoniae of 95.76% to 98.51%.
In some exemplary embodiments, the nucleotide sequence of heat stress protein 70 of mycoplasma ovipneumoniae is set forth in SEQ ID NO:1 is shown.
In order to solve the second technical problem, the invention discloses an application of a mycoplasma ovipneumoniae virulent strain in establishing a mycoplasma ovipneumoniae infection pathogenesis model.
In some embodiments, the virulent strain of Mycoplasma ovipneumoniae is Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) NJ01 strain, which has been deposited at the chinese type culture collection, address: wuhan university, post code 430072, preservation number: CCTCC NO: m2020907. The strain is prepared by the inventor through self-collecting pathological materials in a certain sheep farm in Nanjing in 2018, and separating, purifying and identifying the pathological materials to obtain the mycoplasma ovipneumoniae.
In some embodiments, the heat stress protein 70 of mycoplasma ovipneumoniae has a similarity to other mycoplasma ovipneumoniae of 95.76% to 98.51%.
In some exemplary embodiments, the nucleotide sequence of heat stress protein 70 of mycoplasma ovipneumoniae is set forth in SEQ ID NO:1 is shown.
In some embodiments, the use is for using the mycoplasma ovipneumoniae infection pathogenesis model to study the pathogenic mechanism of mycoplasma ovipneumoniae.
In some embodiments, the use is for the assessment of the efficacy of a mycoplasma ovipneumoniae vaccine using the model for the onset of mycoplasma ovipneumoniae infection.
In some embodiments, the application is to use the mycoplasma ovipneumoniae infection pathogenesis model for evaluation of a medicament against mycoplasma ovipneumoniae infection.
In some embodiments, the mycoplasma ovipneumoniae infection pathogenesis model is established by adopting an attacking preparation of the virulent strain of mycoplasma ovipneumoniae for attacking infection.
In some embodiments, the challenge preparation of the virulent strain of mycoplasma ovipneumoniae is a culture of the virulent strain of mycoplasma ovipneumoniae and/or other challenge preparations prepared from the virulent strain of mycoplasma ovipneumoniae.
In some embodiments, the route of infection is intratracheal injection.
In some more typical embodiments, each sheep is intratracheally injected with 3-6mL of mycoplasma ovipneumoniae culture, observed for clinical symptoms, and examined for lung lesions by autopsy, with typical lung lesions being seen.
In some embodiments, the active ingredient of the virulent strain challenge preparation for mycoplasma ovipneumoniae is the virulent strain of mycoplasma ovipneumoniae described above.
In some embodiments, in the virulent strain challenge preparation of mycoplasma ovipneumoniae, the concentration of the virulent strain of mycoplasma ovipneumoniae is 10 5 CCU/mL or more.
In some embodiments, the mycoplasma ovipneumoniae virulent strain culture is prepared by culturing in a mycoplasma liquid medium.
In some embodiments, the method for culturing the mycoplasma ovipneumoniae virulent strain culture comprises the following steps:
(1) Inoculating a mycoplasma ovipneumoniae NJ01 strain into a mycoplasma liquid culture medium according to the volume ratio of 1-3-1;
(2) Inoculating the first generation culture with mycoplasma liquid culture medium at volume ratio of 1;
(3) And (3) repeating the step (2) for continuous passage, and harvesting when the culture becomes orange yellow to yellow.
In some embodiments, the culturing of step (1) and step (2) is static culturing.
In some embodiments, in step (2), the first generation culture is inoculated into a mycoplasma broth at a volume ratio of 1.
In some embodiments, in step (2), step (2) is repeated for 1-5 generations.
In some embodiments, the mycoplasma liquid culture medium comprises the following components in percentage by weight and is prepared by the following method: eagles liquid 40-50% v/v, hydrolyzed milk protein 0.005-0.02g/mL, phosphate buffer solution of pH 7.4 30-40% v/v, phenol red 0.04g/mL 0.15-0.2% v/v; adjusting pH to 7.4-7.6 with NaOH, autoclaving, adding sterile serum 10% -20%, and fresh yeast extract 1% -2% (v/v) (Zhang Ruiting et al. Improvement of extraction method of yeast extract; preparation of Chinese veterinary drug journal, 1998, method of 02).
In order to solve the third technical problem, the invention discloses an application of a mycoplasma ovipneumoniae virulent strain in preparation of a medicament for treating mycoplasma ovipneumoniae.
In some embodiments, the classification of said virulent strain of Mycoplasma ovipneumoniae is designated as Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) NJ01 strain, which has been deposited with the chinese type culture collection at address: wuhan, wuhan university, zip code 430072, deposit number: CCTCC NO: m2020907.
In some embodiments, the heat stress protein 70 of mycoplasma ovipneumoniae has a similarity to other mycoplasma ovipneumoniae of 95.76% to 98.51%.
In some exemplary embodiments, the nucleotide sequence of heat stress protein 70 of mycoplasma ovipneumoniae is set forth in SEQ ID NO:1 is shown.
In some embodiments, the drug is a vaccine or an antiserum.
In some embodiments, the vaccine is configured from a culture of the virulent strain of mycoplasma ovipneumoniae, each serving comprising the following components: the mycoplasma ovipneumoniae virulent strain NJ01 is not less than 2.86 multiplied by 10 8 CCU, immune adjuvant 5% -75% g/g.
In some embodiments, the immunoadjuvant is white oil for injection, MONTANIDE ISA206VG, ISA 201VG, gel 01ST, ISA 11R, or ISA 760VG, and the like.
In some embodiments, the mycoplasma ovipneumoniae virulent strain culture is prepared by culturing in a mycoplasma liquid medium.
In some embodiments, the method for culturing the mycoplasma ovipneumoniae virulent strain culture comprises the following steps:
(1) Inoculating a mycoplasma ovipneumoniae NJ01 strain into a mycoplasma liquid culture medium according to the volume ratio of 1-3-1;
(2) Inoculating the first generation culture with Mycoplasma liquid culture medium at volume ratio of 1;
(3) And (5) repeating the step (2) for continuous passage, and harvesting when the culture becomes orange yellow to yellow.
In some embodiments, the culturing of step (1) and step (2) is static culturing.
In some embodiments, in step (2), the first generation culture is inoculated into a mycoplasma broth at a volume ratio of 1.
In some embodiments, in step (2), step (2) is repeated for 1-5 generations.
In some embodiments, the mycoplasma liquid culture medium comprises the following components in percentage by weight and is prepared by the following method: eagles liquid 40% -50% v/v, hydrolyzed milk protein 0.005-0.02g/mL, phosphate buffer solution of pH 7.4 30% -40% v/v,0.04g/mL phenol red 0.15% -0.2% v/v; adjusting pH to 7.4-7.6 with NaOH, autoclaving, adding sterile serum 10% -20%, and fresh yeast extract 1% -2% (v/v) (Zhang Ruiting et al. Improvement of extraction method of yeast extract; preparation of Chinese veterinary drug journal, 1998, method of 02).
Has the beneficial effects that: compared with the prior art, the invention has the following advantages:
1) The strain Mycoplasma ovipneumoniae NJ01 provided by the invention has strong toxicity, and can cause morbidity by intratracheal injection of a culture or a preparation.
2) The mycoplasma culture medium adopted for culturing the mycoplasma ovipneumoniae virulent strain is simple and convenient to prepare, low in cost, quick in mycoplasma culture color change and high in titer.
3) The strain mycoplasma ovipneumoniae NJ01 provided by the invention can be recovered through a mycoplasma inoculation liquid culture medium of 1-1 10 CCU/mL, which saves cost and time for preparing the mycoplasma ovipneumoniae attacking preparation and improves efficiency.
4) The mycoplasma ovipneumoniae virulent strain, the application of the mycoplasma ovipneumoniae virulent strain in infection and morbidity tests and the method thereof can better meet the urgent needs of the research on the pathogenic mechanism of the mycoplasma ovipneumoniae, the evaluation on the effectiveness of the vaccine and the screening of pharmaceutical preparations at present, and have wide market prospect and greater economic and social benefits.
5) The medicine for treating mycoplasma ovipneumoniae pneumonia, such as vaccine, prepared by using the virulent strain of mycoplasma ovipneumoniae of the invention has good safety and protection, can meet the urgent need of high-efficiency vaccine in the current market, and has wide market prospect and great economic, social and ecological benefits.
Drawings
The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.
FIG. 1 is a tree of evolutionary trees based on Mycoplasma ovipneumoniae heat stress protein 70; wherein the shaded part is Mycoplasma ovipneumoniae NJ01 strain.
FIG. 2 shows the sheep pneumonia mycoplasma virulent strain NJ01 infecting and attacking sheep lung pathological changes.
Detailed Description
The present invention will now be further described with reference to specific examples, but the invention is not limited thereto.
The following examples are illustrative of the experimental procedures in some embodiments, and are all conventional unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1: acquisition of mycoplasma ovipneumoniae NJ01 strain and culture identification
1. Strain isolation and culture
Screening sheep with suspected mycoplasma ovipneumoniae characteristics in a certain Nanjing sheep farm, collecting diseased sheep with typical mycoplasma ovipneumoniae characteristics, aseptically collecting lungs of diseased sheep, directly injecting mycoplasma liquid culture medium into bronchioles, and sucking to obtain a bronchial lavage fluid. The lavage solution can be directly filtered by a filter with the pore diameter of 0.45 mu m, and then the ratio of 1:1 and 1: diluting the liquid culture medium of the mycoplasma inoculation, carrying out static culture at 37 ℃, carrying out passage when the color of the culture changes from red to orange yellow or yellow, continuously carrying out 5 passages in the way, purifying the solid culture medium of the mycoplasma inoculation after the growth is stable, carrying out cloning purification, inoculating the liquid culture medium of the mycoplasma inoculation, and harvesting the culture after the culture changes to yellow for characteristic identification.
2. Identification of strains
The culture is subjected to Swiss staining microscopy and is in a ring shapeAnd spherical microorganisms, without cell wall and gram-negative. When the culture is cultured in a culture medium containing 0.5 percent of glucose, the color can be changed from red to yellow, which indicates that the fermentation glucose culture medium produces acid. The culture grows well on the mycoplasma liquid culture medium, the culture is cultured for 1 to 5 days at 37 ℃, the pH value of the culture is reduced to 6.6 to 6.8, and the culture is slightly turbid. The concentration of the culture can reach 10 when measured by color change unit 9 CCU/mL. The culture was inoculated with a mycoplasma solid medium and cultured at 37 ℃ for 3 to 7 days, whereupon papillary colonies were observed.
3. Sequencing analysis
PCR was performed with reference to primers for the Mycoplasma ovipneumoniae Total Heat shock protein 70 (Hsp70) gene reported by Bin Zhang, et al molecular characterization of the heat shock protein 70gene in Mycoplasma ovireniae, the Heat bearing Journal 198 (2013) 299-301, and sequencing was performed, the sequence results are shown in SEQ ID NO:1 is shown. The similarity of the sequence and the reported sequence is 95.76 to 98.51 percent by performing Blast comparison, and the result is shown in Table 1. Results of the phylogenetic Tree analysis are shown in FIG. 1, which is a phylogenetic tree based on the nucleotide sequence of heat stress protein 70 of Mycoplasma ovipneumoniae.
TABLE 1 Blast comparison results
4. Strain preservation
The strain is sent to China center for type culture collection for preservation, and the preservation information is as follows: and (3) classification and naming: mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) NJ01 strain; address: wuhan, wuhan university; the preservation date is as follows: 2020, 12 months and 14 days; the preservation number is CCTCC NO: m2020907.
Example 2: mycoplasma ovipneumoniae culture assay
1. Material
Mycoplasma ovipneumoniae NJ01 strain; the preservation number is CCTCC NO: m2020907. Mycoplasma ovipneumoniae IK3-3 strain with the preservation number of CVCC 380.
The mycoplasma culture medium formula and the preparation method are as follows: eagles liquid 50% (v/v), hydrolyzed milk protein 0.02g/mL, phosphate buffer (pH 7.4) 28.8% (v/v), 0.04g/mL phenol red 0.2% (v/v); adjusting pH to 7.4-7.6 with NaOH, autoclaving, adding sterile horse serum 20% (v/v) and fresh yeast extract 1% (v/v). Wherein, the fresh yeast extract refers to Zhang Rutin, etc., the improvement of the extraction method of the yeast extract, china veterinary medicine journal, 1998.
2. Culture test
(1) The strains (mycoplasma ovipneumoniae NJ01 strain and IK3-3 strain) frozen at-20 ℃ are respectively taken out, unfrozen, inoculated in a mycoplasma liquid culture medium in a volume ratio of 1;
(2) Immediately inoculating the first generation culture into a mycoplasma liquid culture medium according to the volume ratio of 1;
(3) After 5 additional passages (different from step (2) in that the inoculation volume ratio was changed to 1 10) according to the method of step (2), the culture was harvested after changing to orange-yellow color, and stored at-20 ℃ or below to obtain cultures of mycoplasma ovipneumoniae NJ01 strain and IK3-3 strain, respectively, and the content of the culture (color change unit, CCU) was measured at the same time.
(4) The 5 or 7-passage culture was inoculated with 6 strains each at a volume ratio of 1 to 10, 3 of which were cultured at 37 ℃ while leaving aside, and the other 3 were cultured in a shaker at 37 ℃ and 160r/min in a constant temperature air bath, and the content of the culture (color change unit, CCU) was measured when the color of the culture became yellow.
3. Results
TABLE 2 Resuscitation, passage, color change time and content of NJ01 strain
| Generation 1 | Generation 2 | Generation 3 | |
5 generation | 6 generation | 7 generation | |
| Inoculation ratio (V/V) | 1:3 | 1:5 | 1:10 | 1:10 | 1:10 | 1:10 | 1:10 |
| Time to change color (h) | 18 | 12 | 12 | 18 | 12 | 10 | 10 |
| CCU content (CCU/mL) | 10 9 | 10 9 | 10 10 | 10 10 | 10 10 | 10 10 | 10 10 |
TABLE 3 IKK 3-3 Resuscitation, passage, color change time and content
| Generation 1 | Generation 2 | Generation 3 | |
5 generation | 6 generation | 7 generation | |
| Inoculation ratio (V/V) | 1:3 | 1:5 | 1:10 | 1:10 | 1:10 | 1:10 | 1:10 |
| Time to change color (h) | 96 | 72 | 72 | 72 | 72 | 72 | 72 |
| CCU content (CCU/mL) | Not testing | 10 7 | 10 8 | 10 8 | 10 8 | 10 8 | 10 8 |
TABLE 4 discoloration time and content of NJ01 strain by standing and shaking culture
As can be seen from the results in the table above, the NJ01 strain can change color after 3-5 generations of resuscitation and passage within 12-18h, and the content can reach 10 10 CCU/mL, higher in growth rate and content than the IK3-3 strain. As can be seen from Table 4, there was no difference in the discoloration time and content between the stationary culture and the shaking culture of the NJ01 strain.
Example 3: infection and pathogenesis test of mycoplasma ovipneumoniae virulent strain
1. Material
The mycoplasma ovipneumoniae NJ01 strain has a preservation number of CCTCC NO: m2020907. The mycoplasma ovipneumoniae IK3-3 strain has a collection number of CVCC 380.
Two mycoplasma cultures were obtained by culturing mycoplasma ovipneumoniae NJ01 strain and IK3-3 strain in a mycoplasma liquid culture medium according to the method described in example 2. The concentration of each strain culture was adjusted to 10 using mycoplasma liquid medium 7 -10 8 CCU/mL as a challenge preparation.
2. Challenge test
Randomly dividing the healthy test goats into 3 groups, wherein each group has 4 goats; first group (NJ 01 strain challenge group) culture with NJ01 strain (10) 7 CCU/mL), and the second group (IK 3-3 challenge group) was challenged with an IK3-3 culture (10) 8 CCU/mL), wherein the toxin counteracting mode is an intratracheal injection mode, and the toxin counteracting dosage is 4mL per goat intratracheal injection. The third group (healthy control group) was intratracheally injected with 4mL of mycoplasma culture medium.
The NJ01 infected sheep showed symptoms such as cough 5-7 days after infection, and severe respiratory symptoms such as sneezing and cough 14d and continued to 28d. After 7 days of infection, the IK3-3 infected group sheep had slight cough, and after 21 days, the respiratory symptoms were most severe and then alleviated. The control group was asymptomatic.
28 days after challenge, all test goats were necropsied and observed for lung lesions, judged according to the mycoplasma hyopneumoniae pneumonopathy index score criteria. The score criteria for mycoplasma ovis pneumonialung lesion index were: after the test goat is subjected to autopsy, the percentage of the average lung area of the lung tissue with the retroversion of pancreas-like flesh on the back and the ventral surface of the left tip leaf, the left heart leaf, the left diaphragm leaf, the right tip leaf, the right heart leaf, the right diaphragm leaf and the accessory leaf to the area of the lung leaf is respectively observed; recording the area ratio of 1% to 25% as 1 minute, 26% to 50% as 2 minutes, 51% to 75% as 3 minutes, and 76% to 100% as 4 minutes; the scores of all lung lobe evaluations are added to obtain the lung disease change index score of the sheep, and the maximum score is 28. The results are shown in table 5 and fig. 2, typical mycoplasma ovipneumoniae pneumonia lesions appear in the lungs of goats inoculated with NJ01 strain cultures, the pulmonary disease lesion index is significantly different from that of a control group (P < 0.05), the morbidity degree is significantly higher than that of IK3-3 strain cultures for virus challenge, the NJ01 strain is stronger in virulence and higher in virulence than that of IK3-3 strains, the NJ01 strain cultures can be used for establishing a mycoplasma ovipneumoniae pneumonia infection pathogenesis model, the model can be used for researching the pathogenic mechanism of the mycoplasma ovipneumoniae, and can also be used for virus challenge in animal infection tests for mycoplasma ovipneumoniae vaccine efficacy evaluation and drug treatment effect evaluation.
In addition, the number of diseased lungs after challenge was counted for each test goat, and the results are shown in table 6. As can be seen from Table 6, 4 lungs of 4 goats have pathological changes after the NJ01 strain is challenged, and the lung disease change index is obviously higher than that of the IK3-3 strain challenged group, which indicates that the NJ01 strain has stronger toxicity and higher toxicity than that of the IK3-3 strain.
TABLE 5 pulmonary lesion index of Mycoplasma capricolum pneumonia after challenge with different strain cultures
| Toxin counteracting toxic strain | Dosage of offensive toxin | Index of lung |
| NJ01 Strain | ||
| 4×10 7 CCU/only | 12±4.1 a | |
| IK3-3 |
4×10 8 CCU/only | 5±3.6 b |
| |
0 CCU/only | 0±0 c |
Note: lung disease index in table 5 is expressed as mean ± standard deviation; a. b and c represent pairwise comparison between each group, the same letters represent no significant difference, and the different letters represent significant difference (p < 0.05).
3. Reisolation of Mycoplasma ovipneumoniae in offending goat diseased lung
The bacterial strain separation and culture method in the embodiment 1 is applied to re-separation and identification of mycoplasma ovipneumoniae from diseased lungs of attacking goats. The PCR identification is carried out by the method for amplifying the heat shock protein 70gene of the Mycoplasma ovis pneumonia reported by Bin Zhang, ethyl molecular characterization of the heat shock protein 70gene in Mycoplasma ovimoniae, the vector Journal 198 (2013) 299-301, and whether each strain can be separated again is examined.
As shown in Table 6, M.ovipneumoniae was isolated from all of the 4 goat diseased lungs challenged with the NJ01 strain, and M.ovipneumoniae was isolated from 3 of the 4 goats challenged with the IK3-3 strain. Test results show that the mycoplasma ovipneumoniae NJ01 strain has strong toxicity and high pathogenicity, so that the lung lesion is obvious, and the re-isolation rate is also high.
TABLE 6 re-isolation of goat mycoplasma pneumoniae after challenge
| Toxin counteracting group | Number of diseased lung | Number of lungs positive for PCR identification | Separation rate of mycoplasma |
| NJ01 Strain | |||
| 4 | 4 | 4/4 | |
| IK3-3 strain | 3 | 3 | 3/4 |
Example 4 preparation of tissue poison counteracting preparation
1. Attack of pathogenic factors
A healthy goat is infected with the virulent strain NJ01 of Mycoplasma ovipneumoniae, the test goat is killed 28 days after the challenge, a lung tissue sample with obvious Mycoplasma ovipneumoniae characteristic lesion in the lung is collected, and PCR, sequencing and identification are carried out by referring to a primer of the Mycoplasma ovipneumoniae Total Heat shock protein 70 (heat shock protein 70, p70) gene reported by Bin Zhang, et al. Sequencing a positive lung tissue sample which accords with the characteristics of the NJ01 strain to prepare a virus attacking preparation for attacking the virus in the efficacy test of the mycoplasma ovipneumoniae vaccine.
2. Tissue poison preparation of poison counteracting preparation
And (2) flushing the lung surface of the lung tissue meeting the tissue virulent preparation requirement by using sterile PBS, draining, shearing the lung tissue into small pieces by using sterilizing scissors, and mixing the small pieces according to the mass ratio of 1:4 mixing the lung tissue with Hank's solution, placing the mixture into a sterilized homogenizer for homogenization, filtering and collecting filtrate. Mixing the filtrate with a lyoprotectant (such as an aqueous solution containing 1.5% (w/v) gelatin and 12.5% (w/v) sucrose) at a volume ratio of 1.5:1, fully and uniformly mixing, adding penicillin and streptomycin with final concentration of 1000 units/mL, and fully and uniformly mixing to obtain the tissue virulent strains for counteracting the toxin of the NJ01 strain. Aseptically adding the tissue virulent suspension for counteracting the virus into a sterilization bottle, freeze-drying to obtain tissue virulent freeze-dried substances of each strain, checking that the freeze-dried physical properties are qualified, marking, and storing at-20 ℃ for later use.
Example 5: preparation of mycoplasma ovipneumoniae inactivated vaccine
1. Material
The mycoplasma ovipneumoniae NJ01 strain has a preservation number of CCTCC NO: m2020907. Esomarcol 52 white oil.
2. Preparing seedling
2.1 antigen preparation and inactivation
The Mycoplasma ovipneumoniae NJ01 strain was cultured in a Mycoplasma liquid medium according to the method described in example 2, to obtain a Mycoplasma 5 culture. Pure inspection and content measurement were performed according to the specifications of the Chinese veterinary pharmacopoeia.
Adding the qualified bacterial liquid for preparing the seedlings into thimerosal with the final concentration of 0.005 percent, and inactivating the thimerosal at 37 ℃. And after inactivation and inspection, the obtained product is used for seedling preparation.
2.2 preparing the seedlings
Each 1mL portion contains 2.86X 10 inactivated Mycoplasma ovipneumoniae 8 CCU, the proportion of the vaccine is determined by the adjuvant (the weight percentage of the immunologic adjuvant in the vaccine is 5% -75%), and the antigen is supplemented by PBS.
2.2.1 preparation and mixing of white oil for injection
Taking 94 parts (by weight) of white oil for injection and span-80 parts, adding the white oil for injection into an oil phase tank, uniformly stirring, sterilizing at 121 ℃ for 30 minutes, and cooling to room temperature for later use. And (3) adding 96 parts of bacterium liquid qualified in inactivation test and 4 parts of tween-80 into the water phase tank, and stirring and uniformly mixing for later use. Adding 2-3 parts of oil phase into an emulsification tank, firstly stirring at low speed for 30 minutes, then slowly adding 1 part of water phase, continuously stirring uniformly, and emulsifying by using a high-speed shearing machine to prepare the water-in-oil type emulsion.
2.2.2ISA 206VG (SEPPIC), ISA 201VG (SEPPIC), gel 01ST (SEPPIC), ISA 11R VG (SEPPIC) and their mixture
Preparing seedlings and mixing according to the instruction. As ISA 201VG, 50:50 preparing the vaccine, preheating the adjuvant to 31 ℃, stirring with low shearing force, maintaining at 30 ℃, adding the antigen (water phase) into the adjuvant, and uniformly stirring. As ISA 11R VG, in a weight ratio of adjuvant to antigen of 15:85, adding the antigen water phase into the adjuvant at room temperature or lower, and uniformly stirring by using a low-shear stirrer. For example, gel 01ST, adjuvant is prepared by mixing 10% of adjuvant, adding antigen water phase into adjuvant at room temperature or lower, and stirring with low shear stirrer.
3. Inspection of finished product
3.1 Properties
The dosage form should be suitable for the application. The vaccine prepared from white oil for injection is water-in-oil type, the vaccine prepared from ISA 201VG is oil-in-water type, the vaccine prepared from ISA 11R VG is oil-in-water type, and the vaccine prepared from Gel 01ST is water dosage form.
3.2 stability
10mL of vaccine is sucked and added into a centrifuge tube, and the centrifuge tube is centrifuged at 3000r/min for 15 minutes, and the water phase separated out from the tube bottom does not exceed 0.5mL.
3.3 viscosity
The test is carried out according to the appendix of the current Chinese beast pharmacopoeia, and the test does not exceed 200cP.
3.4 sterility testing
The bacteria-free growth is carried out according to the examination of the appendix of the current Chinese beast pharmacopoeia.
Example 6: immunity test of sheep mycoplasma pneumoniae inactivated vaccine
1. Material
Vaccine to be tested: mycoplasma ovipneumoniae inactivated vaccine (NJ 01 strain) oil adjuvant (each head contains Mycoplasma ovipneumoniae NJ01 strain 2.86 multiplied by 10) 8 CCU), prepared according to the method of example 5.
The similar vaccine comprises: inactivated vaccine for mycoplasma capricolum pneumonia (MoGH 3-3 strain + M87-1 strain) produced by a certain company in China (each head contains mycoplasma ovipneumoniae MoGH3-3 strain 4 x 10 8 CCU)。
Mycoplasma ovipneumoniae strain tissue is virulent and prepared according to the method of example 4.
15 healthy susceptible sheep of 3 months of age.
2. Design of experiments
Vaccine immunization group: immunizing sheep with the vaccine to be tested: 5 sheep were immunized with the vaccine to be tested, 1mL each.
Vaccine immunization group: similar vaccines are used to immunize sheep: the same vaccine was injected subcutaneously with 3mL of vaccine per one vaccine, according to the vaccine instructions.
Meanwhile, 5 sheep with the same conditions are set as a challenge control group and a healthy control group respectively.
Clinical responses were observed within 28 days after vaccine immunization. 35 days after the initial immunization, the vaccine immunization group and the challenge control group are subjected to a challenge test, 5mL of mycoplasma ovipneumoniae strain prepared in example 4 is injected into each sheep intratracheally, the autopsy is carried out 28 days after the challenge, the lung lesion score is counted, and the lung lesion index reduction rate is calculated.
3. Observation of clinical response
The test sheep are observed for 28 days after the vaccine is injected, and as a result, no adverse reaction is caused to the sheep immunized by the vaccine to be tested, 2 sheep immunized by the similar vaccine have lassitude after the immunization, but the sheep are recovered to be normal in three days after the immunization. No other abnormal clinical response was seen.
4. Comparative efficacy test
The experimental sheep challenge is observed in 28 days, 5 sheep in a challenge control group have cough with different degrees, the sheep immunized by the vaccine to be tested have no characteristic symptoms of mycoplasma pneumoniae such as cough, and 2 sheep immunized by the similar vaccine have characteristic symptoms of mycoplasma pneumoniae such as slight cough.
After 28 days of virus challenge, the examination is carried out, lung lesions are observed, and 5 sheep in a virus challenge control group have typical lesions as a result, which shows that the virus challenge preparation prepared in example 4 can cause diseases and can be used for tests such as the immunity efficacy of the mycoplasma ovipneumoniae inactivated vaccine and the like; no typical lesion appears in an immune group of the mycoplasma ovipneumoniae inactivated vaccine (NJ 01 strain) to be tested, and 2 of the immune groups of the similar vaccines only have slight lesions.
Example 7 preparation of Mycoplasma ovipneumoniae antiserum and Metabolic inhibition assay
1. Material
Mycoplasma ovipneumoniae NJ01 strain; the preservation number is CCTCC NO: m2020907. Mycoplasma ovipneumoniae strain Y98, reference strain.
The mycoplasma culture medium formula and the preparation method are as follows: eagles liquid 50% (v/v), hydrolyzed milk protein 0.02g/mL, phosphate buffer (pH 7.4) 28.8% (v/v), 0.04g/mL phenol red 0.2% (v/v); adjusting pH to 7.4-7.6 with NaOH, autoclaving, adding sterile horse serum 20% (v/v) and fresh yeast extract 1% (v/v). Wherein, the fresh yeast extract is prepared by referring to Zhang Ting and the like, the improvement of the extraction method of the yeast extract, china veterinary medicine journal, 1998, no. 02.
Clean grade New Zealand white rabbits, jinling rabbit farm.
2. Preparation of rabbit anti-sheep mycoplasma pneumonia hyperimmune serum
2.1 preparation of immunogens
(1) Inoculating mycoplasma ovipneumoniae NJ01 strain into mycoplasma liquid culture medium, culturing at 37 deg.C, rejuvenating and enlarging culturing, collecting culture when the culture medium turns yellow (pH 6.6-7.0), and determining CCU content.
(2) And (2) centrifuging the culture obtained in the step (1) at a high speed, washing the obtained precipitate which is concentrated thalli with PBS, concentrating and washing the thalli, carrying out ultrasonic disruption, and measuring the protein content, wherein the protein content is more than 3mg/mL and is used as a protein antigen.
(3) And (3) carrying out sterile emulsification on the protein prepared in the step (2) with Freund's complete adjuvant and Freund's incomplete adjuvant according to the specification to prepare a Freund's complete adjuvant preparation of the mycoplasma ovipneumoniae NJ01 strain and a Freund's incomplete adjuvant preparation of the mycoplasma ovipneumoniae NJ01 strain.
(4) According to the steps (1) to (3), a Freund's complete adjuvant preparation of Mycoplasma ovipneumoniae Y98 strain and a Freund's incomplete adjuvant preparation of Mycoplasma ovipneumoniae Y98 strain were prepared.
2.2 preparation of hyperimmune serum
On the day of first immunization, 1mL of mycoplasma ovipneumoniae NJ01 strain Freund complete adjuvant preparation is injected subcutaneously at multiple points on the back of a New Zealand white rabbit; injecting 1mL of Mycoplasma ovipneumoniae NJ01 strain Freund incomplete adjuvant preparation into the backs of rabbits at multiple subcutaneous points 14 days after the first immunization; on days 21, 28 and 35 after priming, 1mL of protein antigen was injected intramuscularly in the rabbit leg, respectively. And (3) collecting blood 42 days after the first immunization, separating serum, performing sterile filtration, and preparing the rabbit anti-mycoplasma ovipneumoniae NJ01 strain hyperimmune serum after inspection.
The rabbit anti-mycoplasma ovipneumoniae Y98 strain hyperimmune serum is prepared according to the method.
3. Rabbit anti-sheep mycoplasma pneumonia hyperimmune serum detection test
The reference literature (research on the metabolic inhibition of mycoplasma hyopneumoniae by hyperimmune serum of different animals such as Sunyibop, etc. the method is carried out by the method of 2016, 50 (12): 7-11 in Chinese veterinary medicine journal.
The mycoplasma ovipneumoniae culture is diluted by 10 times to 10 -10 (specific dilution concentrations are shown in Table 7); the rabbit anti-mycoplasma ovipneumoniae hyperimmune serum is diluted to 1/10 by using a mycoplasma liquid culture medium, namely the rabbit anti-mycoplasma ovipneumoniae hyperimmune serum accounts for 10% of the total volume of the rabbit anti-mycoplasma ovipneumoniae hyperimmune serum and the mycoplasma liquid culture medium.
Adding the diluted mycoplasma ovipneumoniae cultures into the diluted mycoplasma ovipneumoniae antiserum with the same volume as the diluted mycoplasma ovipneumoniae culture, uniformly mixing, using as a test group, and culturing at 37 ℃; meanwhile, the content of CCU is measured by the mycoplasma ovipneumoniae culture; additionally arranging mycoplasma liquid culture medium as culture medium control group, and standing and culturing at 37 deg.C; and judging the result when no new color change is generated within 2 days. The blank control should not change color, and the anti-mycoplasma ovipneumoniae metabolism inhibition value (serum color change dilution/CCU color change dilution) of the antiserum can be judged according to the CCU content measurement result and the color change condition of the test group.
4. Results
The rabbit anti-sheep mycoplasma pneumonia hyper-immune serum is tested and cultured at 37 ℃, and the color change condition is shown in table 6.
TABLE 7 determination results of metabolic inhibition value of rabbit anti-mycoplasma ovipneumoniae hyperimmune serum against mycoplasma ovipneumoniae
As can be seen from the results, cultivationThe nutrient control group has no discoloration, and the content of NJ01 strain is 10 9 Measurement of antiserum group species 10 with CCU/mL, NJ01 Strain -1 -10 -6 Color change by dilution, 10 -7 -10 -9 The dilution is not discolored, and the metabolic inhibition value of the rabbit anti-mycoplasma ovipneumoniae hyperimmune serum is 10 3 . The content of Y98 strain is 10 8 CCU/mL, the test is established; determination of antiserum 10 against strain Y98 -1 -10 -5 Color change by dilution, 10 -6 -10 -8 The dilution is not discolored, and the metabolic inhibition value of the rabbit anti-mycoplasma ovipneumoniae hyperimmune serum is 10 3 . Therefore, the results of the mycoplasma ovipneumoniae NJ01 strain and the Y98 strain on the determination of the metabolic inhibition value of rabbit anti-mycoplasma ovipneumoniae hyperimmune serum are the same, the NJ01 strain content is higher than that of the Y98 strain, and the time for stopping the color change of the culture is also earlier than that of the Y98 strain. Therefore, the NJ01 strain is accurately and timely used for evaluating the inhibiting effect of the preparation such as the serum on the mycoplasma ovipneumoniae and the like.
The invention provides a virulent strain of mycoplasma ovipneumoniae and a thought and a method for application thereof in infection pathogenesis and preparation of medicines for treating mycoplasma ovipneumoniae, and a plurality of methods and ways for realizing the technical scheme are provided. All the components not specified in the present embodiment can be realized by the prior art.
Sequence listing
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<120> mycoplasma ovipneumoniae virulent strain and application thereof
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gttatcacag ttcctgctta ttttgataat gctcaacgtg aagcgacaaa aaatgccgga 420
aaaatcgcag gattagaagt tgaaagaata attaacgaac caacagctgc cgcacttgca 480
tttggtcttg acaaaaccga aaaagaaatg aaagtgcttg tttatgactt aggtggtgga 540
acttttgacg tttcagttct tgaattatct aacggaactt ttgaagtttt atcaacaagt 600
ggtgataacc atttaggtgg tgatgactgg gataatcaaa ttgttgactg aatggttaaa 660
agaattaaag aagagtacga ttttgatgca aaaagtgaca aaatggcact tactcgtcta 720
aaagaagaag ctgaaaaaac caaaattaac ctatcaaatc aaagtgtttc aacaatttcc 780
ttaccatttt taggtctagg aaaaaatggt ccaattaacg ttgaacttga actaaaaaga 840
tcagactttg aaaaaatgac agctcaccta attgaccgaa caagaaaacc aatcgttgat 900
gccttaaaac aagcaaaaat tgaagcaagt gagcttgacg aggttcttct tgttggtgga 960
tcaacacgta tgccagctgt tcagacaatg attgaacaca ctttaaataa aaagccaaat 1020
cgttcaataa atcctgatga agttgtggca attggagctg caattcaagg tggagttctt 1080
gctggagaaa ttagtgatgt tttattactt gacgttactc ctttaacttt aggaattgaa 1140
accttaggtg gagtttcaac cccgttaatt ccaagaaata caacaattcc ggtaacaaaa 1200
tcacaaattt tctcaacagc tgaagacaat caaaccgaag ttacaatttc tgtagtccaa 1260
ggtgaaagac aacttgctgc tgataacaaa atgttaggtc gctttaatct ttcaggaatc 1320
gagcctgctc ctcgtggtct tccacaaatt gaagttagtt tttcaattga cgtcaacggt 1380
attacaactg tttcagcaaa agataaaaaa actggaaaag aacaaacaat cacaattaaa 1440
aatacttcaa ctttatctga agaagaaatt aacagaatga ttcaagaagc cgaagaaaat 1500
cgtgaagctg atgcaatcaa aaaagataaa attgaaacaa cagttcgtgc cgaaggtctt 1560
attaatcaac ttgaaaaatc aataactgat caaggtgaaa aaattgatcc taaacaaaaa 1620
gaacttcttg aaaaacaaat tcaagaactc aaagatcttt taaaagaaga aaaaattgac 1680
gaactaaaaa caaaattaga ccaaattgag caagcagccc aagcttttgc ccaagcaagc 1740
gctcaacaag ctaacagtgc aagtgaaact gattcagatt caaacacaat tgatgctgaa 1800
atcaaacaaa attaa 1815
Claims (11)
1. The Mycoplasma ovipneumoniae virulent strain is characterized in that the Mycoplasma ovipneumoniae is classified and named as Mycoplasma ovipneumoniae (Mycoplasma ovipneumoniae) NJ01 strain, is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2020907, the preservation date is 12 months and 14 days in 2020.
2. The application of the mycoplasma ovipneumoniae virulent strain in establishing a mycoplasma ovipneumoniae infection pathogenesis model, wherein the mycoplasma ovipneumoniae virulent strain is the mycoplasma ovipneumoniae virulent strain in claim 1.
3. Use according to claim 2, wherein the model of the onset of mycoplasma ovipneumoniae infection is used to study the pathogenesis of mycoplasma ovipneumoniae infection or to assess the efficacy of a mycoplasma ovipneumoniae vaccine.
4. The use according to claim 2, wherein the model of the onset of mycoplasma ovipneumoniae infection is used for the evaluation of a non-therapeutic drug against mycoplasma ovipneumoniae infection.
5. The use according to claim 2, wherein the model of the mycoplasma ovipneumoniae infection is established by using a virus attacking preparation of the virulent strain of mycoplasma ovipneumoniae to carry out virus attacking infection; wherein the toxin attacking preparation of the mycoplasma ovipneumoniae virulent strain is a culture of the mycoplasma ovipneumoniae virulent strain and/or other toxin attacking preparations prepared from the mycoplasma ovipneumoniae virulent strain; wherein the infection is achieved by intratracheal injection.
6. The use of claim 5, wherein the virulent strain of Mycoplasma ovipneumoniae is present in the challenge formulation at a concentration of 10 5 CCU/mL or more.
7. The application of the mycoplasma ovipneumoniae virulent strain in preparing a medicine for treating mycoplasma ovipneumoniae is characterized in that the mycoplasma ovipneumoniae virulent strain is the mycoplasma ovipneumoniae virulent strain in claim 1.
8. The use of claim 7, wherein the medicament is a vaccine or an antiserum.
9. The use according to claim 8, wherein the vaccine is formulated from a culture of a virulent strain of Mycoplasma ovipneumoniae, each serving comprising the following components: the mycoplasma ovipneumoniae virulent strain NJ01 is not less than 2.86 multiplied by 10 8 CCU and immunologic adjuvant 5% -75% g/g.
10. The use according to claim 9, wherein the method for culturing the culture of the virulent strain of mycoplasma ovipneumoniae comprises the following steps:
(1) Inoculating a mycoplasma ovipneumoniae NJ01 strain into a mycoplasma liquid culture medium according to the volume ratio of 1-3-1;
(2) Inoculating the first generation culture with mycoplasma liquid culture medium at volume ratio of 1;
(3) Repeating the step (2) for continuous passage, and harvesting when the culture becomes orange yellow-yellow.
11. The use of claim 10, wherein the mycoplasma liquid culture medium comprises the following components by weight percent: 40-50% v/v of Eagles liquid, 0.005-0.02g/mL of hydrolyzed milk protein, 30-40% v/v of phosphate buffer solution with pH of 7.4 and 0.15-0.2% v/v of phenol red with the concentration of 0.04 g/mL; adjusting pH value to 7.4-7.6 with NaOH, autoclaving, adding 10-20% v/v sterile serum and 1-2% v/v fresh yeast extract.
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| CN110452855A (en) * | 2019-09-04 | 2019-11-15 | 中国农业科学院兰州兽医研究所 | A kind of preparation method of serum-free mycoplasma ovine pneumoniae antigen |
| CN111690554A (en) * | 2020-05-23 | 2020-09-22 | 内蒙古自治区农牧业科学院 | Combined strain for preparing mycoplasma ovis pneumonia vaccine, mycoplasma ovis pneumonia trivalent inactivated vaccine and preparation method thereof |
| CN112481154A (en) * | 2020-11-18 | 2021-03-12 | 内蒙古大学 | Mycoplasma ovipneumoniae vaccine strain, vaccine composition prepared from vaccine strain and application of vaccine composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110452855A (en) * | 2019-09-04 | 2019-11-15 | 中国农业科学院兰州兽医研究所 | A kind of preparation method of serum-free mycoplasma ovine pneumoniae antigen |
| CN111690554A (en) * | 2020-05-23 | 2020-09-22 | 内蒙古自治区农牧业科学院 | Combined strain for preparing mycoplasma ovis pneumonia vaccine, mycoplasma ovis pneumonia trivalent inactivated vaccine and preparation method thereof |
| CN112481154A (en) * | 2020-11-18 | 2021-03-12 | 内蒙古大学 | Mycoplasma ovipneumoniae vaccine strain, vaccine composition prepared from vaccine strain and application of vaccine composition |
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Application publication date: 20210608 Assignee: Tiankang biopharmaceutical Co.,Ltd. Assignor: JIANGSU ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2023980048777 Denomination of invention: A highly virulent strain of Mycoplasma sheep pneumonia and its application Granted publication date: 20230124 License type: Common License Record date: 20231201 |